title: Dual LC-MS platform for high-throughput proteome analysis.
abstract: We describe a dual-column interface for parallel chromatography to improve throughput during LC-MS experimentation. The system employs a high-voltage switch to operate two capillary column/nanospray emitters fixed at the MS orifice. Sequentially loading one column while operating the second nearly doubles the LC-MS duty cycle. Given the innate run-to-run variation of a nanospray LC-MS (12% RSD peak area; 2% retention time), the intercolumn variability of the platform showed no meaningful difference for proteome analysis, with equal numbers of proteins and peptides identified per column. Applied to GeLC analysis of an E. coli extract, throughput was increased using one of three methods: doubling the number of replicates, increasing the LC gradient length, or sectioning the gel into twice as many fractions. Each method increased the total number of identifications as well as detection throughput (number of peptides/proteins identified per hour). The greatest improvement was achieved by doubling the number of gel slices (10 vs 5). Analysis on the dual column platform provided a 26% increase in peptides identified per hour (24% proteins). This translates into ~50% more total proteins and peptides identified in the experiment using the dual LC-MS platform.
authors.affiliation: Department of Pathology, Dalhousie University , 11th Floor Tupper Medical Building, Room 11B, Halifax, NS B3H 4R2, Canada.
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Orton
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Wall
authors.forename: Alan A
authors.initials: AA
authors.lastname: Doucette
mesh_terms: D053801:Capillary Electrochromatography / Q000295:instrumentation*; D002853:Chromatography, Liquid / Q000295:instrumentation*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis*; D057166:High-Throughput Screening Assays; D013058:Mass Spectrometry / Q000295:instrumentation*; D059748:Proteolysis; D020543:Proteome / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation*
chemical_list: D029968:Escherichia coli Proteins; D020543:Proteome